The Tissue Culture Core Laboratory will be responsible for providing investigators with cultured cells, antibodies, and facilities for routine microscopic analysis of biological samples. The core will be directed by Dr. Goldstein. He will be assisted by Drs. Y.K. Ho, Richard Anderson, Joachim Herz, and Rob Rawson. The technical work in the Tissue Culture Facility of this Core is carried out by four experienced technicians (Lisa Beatty, Angela Carroll, Jill Fairless, Marissa Hodgin) and one laboratory assistant (Rose Marshall). The Tissue Culture Facility of this Core is located in the Department of Molecular Genetics and consists of three suites of rooms that are used solely for tissue culture. One suite contains four work modules that open into a common work area; the second suite contains three work modules that open into a common work area; and the third suite contains two work modules, one for adenovirus work and the other for baculovirus. Each module is equipped with a sterile work area (Biological Safety Cabinet hood), one or more CO2 incubators, a refrigerator, and an inverted microscope. The common work area in each of the suites contains one or two sterile work areas. The entire facility contains a total of 12 inverted microscopes (6 of which are phase-contrast microscopes), 1 stereo microscope, 15 sterile work areas (hoods), 28 singlechamber CO2 incubators, 2 non-CO2 refrigerated incubators, 1 roller bottle incubator, 3 refrigerated incubator shakers, 3 table-top refrigerated centrifuges, and 9 refrigerators. Four liquid nitrogen freezers for storage of cell lines are located in the common work area adjacent to the work modules. In addition to this standard equipment, we have a Zeiss Axiovert 35 M inverted fluorescence microscope that allows us to visualize cells directly in the Petri dish (under sterile conditions) so as to determine whether or not they have taken up reconstituted fluorescent LDL. This method is the assay of choice for identifying and monitoring mutant cholesterol auxotrophic cell clones that are resistant to amphotericin and thus fail to take up fluorescent LDL (Research Project 1). The success of the entire Program Project depends on the smooth operation of the Tissue Culture Facility. Each of the six Research Projects will depend directly on the availability of cultured cells. Over the past 33 years, we have developed considerable experience in maintaining and growing multiple cell types, including diploid human fibroblasts, Chinese hamster ovary (CHO) cells, Sp2/O-Ag14 myeloma cells, simian COS cells, human embryonic kidney (HEK)-293 cells, rat-1 fibroblasts, mouse embryonic fibroblasts, human lymphoblastoid cells, HeLa cells, rat FT02B hepatoma cells, human Huh-7 hepatoma cells, SV-40 transformed fibroblasts (SV-589), Sf9 insect cells, High Five insect cells, and Drosophila S2 cells. We have also become expert in culturing pluripotent mouse embryonic stem cells (ES cells), which will be used in Research Projects 2, 3, 5, and 6 for gene targeting experiments. Since 1973, over 1000 different primary human fibroblast cell strains, derived from skin biopsies from normal subjects as well as from patients with metabolic disorders, have been initiated in our laboratory. Multiple aliquots of cells from early passages of each of these strains are stored in liquid nitrogen. Our collection of frozen mutant cell lines includes fibroblasts from more than 160 subjects with the homozygous form of familial hypercholesterolemia (FH). Remarkably, we have been successful in carrying out metabolic experiments on mutant fibroblasts that we froze away 32 years ago. Since 1984, thousands of transfection experiments have been carried out in which we have introduced into various cell lines multiple plasmid constructs containing either the protein-coding region or the promoter regions of the genes for the LDL receptor, HMG CoA reductase, HMG CoA synthase, the oxysterol binding protein, SREBP-1 and -2, SCAP, Site-1 protease, Site-2 protease, lnsig-1 and -2, and.a and psubunits of farnesyltransferasel and a and ft-subunits of Rab geranylgeranyl transferase, REP-1 and -2, cholesterol 7-hydroxylase, oxysterol 7-hydroxylase, VLDL receptor, various domains of LRP, RAP, ApoER2, SR-B1 scavenger receptor, ARM, ABCG5/8, PCSK9, etc. From these transfections, more than 1200 stable and permanent cell lines have been established and used for biochemical analyses. Multiple aliquots of each of these transfected cell lines have been frozen in liquid nitrogen. These numbers do not include the thousands of transient transfections performed in HEK-293, COS, CHO cells, and SV589 cells. Based on current experience, we anticipate that we will be setting up for experiments approximately 600 Petri dishes of cells each day, of which 350 will be small dishes (60-mm) and 250 will be large dishes (100-mm). We have learned that certain aspects of quality control are essential in obtaining reproducible results for metabolic experiments in tissue culture cells. The following measures are rigorously enforced in our dayto- day operation: 1) An aliquot of calf serum from each batch of 100 liters is pretested prior to its general use so as to ensure that the serum is not toxic to our fibroblasts and that it supports a high cloning efficiency for CHO cells. 2) Cells in current use are routinely checked for Mycoplasma contamination in two ways: 1) by sending aliquots of the medium from 10 different cell lines to the ATCC Mycoplasma Testing Service (Manassas, VA) once per year; and 2) by our own testing with the use of a Myoplasma PCR Elisa test kit (Roche). 3) The generation time of each human fibroblast cell strain is monitored by a standard procedure, as follows: each week, each confluent stock flask is split 1:2, and the passage number is raised by 2. All stock flasks are discarded when the passage number exceeds 25. This procedure ensures that the growth rates of different aliquots of the same cell strain do not differ appreciably, and it also ensures that our experiments are carried out on cells that are well within Phase II of their growth cycle (Littlefield, J.W. In Variation, Senescence, and Neoplasia in Cultured Somatic Cells. Harvard University Press, Boston, 1976. pp. 54-77). 4) In setting up a typical experiment with nontransfected or permanently transfected cells, cells from the stock flasks are trypsinized under rigidly controlled conditions, counted using a hemacytometer, and 10 to 30 replicate dishes of cells are prepared under identical conditions. 5) In experiments designed to compare a normal and a mutant cell line or a nontransfected and a transfected cell line, Petri dishes of cells from both cell lines are always set up in parallel on the same day and grown in the same medium side by side in the same incubator. With these precautions, among others, our laboratory has been able to generate data on fibroblasts and other cell types that are reproducible from year to year, thus enabling rapid progress in the delineation of metabolic processes. In addition to the maintenance of stock cell lines and preparation of cultured cells for experiments, the Tissue Culture Core will be involved in the following activities: 1) Maintenance of our previously characterized hybridoma cell lines in suspension culture (in roller bottles) and production of large amounts of IgG fractions of monoclonal antibodies from culture medium. The following monoclonal antibodies are currently being produced and purified by Protein G column chromatography for use by investigators in the Program Project: anti-LDL receptor (lgG-C7); anti-SREBP-1 and anti-SREBP-2 (lgG-2A4, lgG-7D4, lgG-1C6, lgG-1D2); anti-Scap (lgG-9D5); anti-HMG CoA reductase (lgG-A9); and anti-Myc (lgG-9E10). Several new monoclonal antibodies against the membrane domain of Scap and against different domains of PCSK9 are currently being characterized. Moreover, mice have recently been injected with purified human lnsig-1 and lnsig-2 for producing hybridomas. 2) Purification by Protein G chromatography of more than 20 different polyclonal rabbit antibodies directed against the multiple proteins that are studied in this Program Project Grant. 3) Growth of large volumes of HEK-293S cells in suspension culture that allow the efficient transfection of cDNAs and the production of their encoded proteins. The HEK-293S cells have proved extremely valuable in the initial evaluation of the functional properties of proteins produced by newly cloned cDNAs. 4) Isolating, maintaining, and freezing away cloned cell lines that have been transfected with mutated versions of various cDNA and promoter constructs